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Applied Radiation and Isotopes 67 (2009) 1622–1628

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Applied Radiation and Isotopes


journal homepage: www.elsevier.com/locate/apradiso

Effects of g-radiation on the fungus Alternaria alternata in artificially


inoculated cereal samples
R. Braghini a,, C.R. Pozzi b, S. Aquino c, L.O. Rocha a, B. Corrêa a
a
Departamento de Microbiologia, Instituto de Ciências Biomédicas II, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374, CEP 05508-900 São Paulo, Brazil
b
Instituto de Zootecnia, Rua Heitor Penteado 56, CEP 13460-000, Nova Odessa, São Paulo, Brazil
c
Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 , CEP 01246-902 ,São Paulo, Brazil

a r t i c l e in f o a b s t r a c t

Article history: The objective of this study was to evaluate the effects of different g-radiation doses on the growth of
Received 13 August 2008 Alternaria alternata in artificially inoculated cereal samples. Seeds and grains were divided into four
Received in revised form groups: Control Group (not irradiated), and Groups 1, 2 and 3, inoculated with an A. alternata spore
9 March 2009
suspension (1 106 spores/mL) and exposed to 2, 5 and 10 kGy, respectively. Serial dilutions of the
Accepted 9 March 2009
samples were prepared and seeded on DRBC (dichloran rose bengal chloramphenicol agar) and DCMA
(dichloran chloramphenicol malt extract agar) media, after which the number of colony-forming units
Keywords: per gram was determined in each group. In addition, fungal morphology after irradiation was analyzed
Foods by scanning electron microscopy (SEM). The results showed that ionizing radiation at a dose of 5 kGy
Cereals
was effective in reducing the growth of A. alternata. However, a dose of 10 kGy was necessary to inhibit
Alternaria alternata
fungal growth completely. SEM made it possible to visualize structural alterations induced by the
g-radiation
Fungi different g-radiation doses used.
& 2009 Elsevier Ltd. All rights reserved.

1. Introduction being the most studied (Combe et al., 1970; Pero et al., 1973;
Rosett et al., 1957; Visconti et al., 1986). Cereal grains are
Fungi can contaminate foods from cultivation to harvest, frequently contaminated with various Alternaria species, particu-
during transportation and storage, and in various production larly A. alternata (Conner and Thomas, 1985). They occur naturally
phases, whenever the fungus is under favorable conditions of in sunflower seeds (Dalcero et al., 1997; Pozzi et al., 2005), wheat
temperature and humidity (Frisvad and Samson, 1991). The effects (Li et al., 2001; Logrieco et al., 1990; Aziz et al., 2006), corn (Aziz
of fungal invasion include a reduced germination potential, et al., 2006; Torres et al., 1998), and rice (Broggi et al., 2007; Tonon
development of visible moldiness, discoloration, unpleasant odor, et al., 1997), among others.
loss of dry matter, heating, chemical and nutritional changes, Irradiation has been used to preserve foods and to produce
loss of quality, and production of mycotoxins (Christensen and foods free of pathogenic microorganisms (Rustom, 1997). Irradia-
Kaufmann, 1969). tion inactivates microorganisms that decompose foods, particu-
Mycotoxins are a group of toxic substances produced by larly bacteria, molds and yeast. This treatment also destroys
filamentous fungi, which, depending on their concentrations in pathogenic organisms, including worms and insects, which
foods and feeds, may pose serious problems to human and animal degrade the quality of stored foods (OMS, 1989). Ionizing radiation
health (Moss, 1998). has been widely recognized as a method of decontamination
Species of the genus Alternaria are abundant in nature. These of foodstuffs. Many reviews have summarized the nutritional
fungal species invade cereals, oleaginous plants and other crops. adequacy of irradiated foods. They clearly demonstrate that
They are able to produce a wide variety of mycotoxins under irradiation results in minimal, if at all noticeable, changes in the
favorable conditions of temperature and humidity (Chulze et al., taste, provided that the optimal dose for each type of food is not
1995). The genus Alternaria produces about 71 known mycotoxins exceeded (Diehl and Josephson, 1994). In general, irradiation
(Montemurro and Visconti, 1992). A. alternata, the most toxigenic to the recommended doses changes the chemical composition of
species of the genus, is known to produce seven toxins in foods, foods very little. According to Diehl (1992, 1995), at doses below
with alternariol (AOH) and alternariol monomethyl ether (AME) 1 kGy, nutritional losses are considered to be insignificant, and
none of the chemical changes found in irradiated foods is harmful,
dangerous or even lying outside of the limits normally observed
 Corresponding author. Tel.: +55 11 30917295; fax: +55 11 30917354. (Satin, 1993; Delincée et al., 1998). Aziz et al. (2006) concluded
E-mail address: raquelbraghini@yahoo.com.br (R. Braghini). that doses of up to 10 kGy are highly effective in microbial

0969-8043/$ - see front matter & 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apradiso.2009.03.004
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decontamination and have no adverse effects on the nutritional Spores were counted in a Neubauer chamber, and the
quality of cereal grains (see also World Health Organization, concentration of the final solution was adjusted to 1 106
1994). spores/mL, according to the paper by Aziz et al. (1991).
In view of these facts, we have made an attempt to evaluate the
effects of a range of g-radiation doses on the growth of Alternaria 2.6. Inoculation of the spore suspension into cereal samples
alternata in artificially inoculated cereal samples.
The cereal samples were inoculated with 1-mL portions of the
A. alternata suspension containing 1 106 spores/mL. The inocu-
2. Materials and methods lated samples were stored in a plastic container sealed with
adhesive tape at 25 1C for 21 days in a BOD incubator, with
2.1. Cereal samples humidity and temperature controlled with a thermohygrometer.
After this period, samples of Groups 1, 2 and 3 were irradiated to
Samples of sunflower (Catissol 1), corn (Agromen 2012 2, 5 and 10 kGy, respectively. Samples of the control group were
Hybrid), rice (BRS Atlanta), and wheat (IAC 370) were used. not irradiated.

2.7. Determination of the fungal mycoflora in the control group and


2.2. Determination of the natural fungal microflora in seeds and
irradiated samples
grains

After incubation, the samples were triturated, irradiated, and


The samples were triturated, and 10 g aliquots of each sample
10 g aliquots of each sample were transferred to an Erlenmeyer
were transferred to Erlenmeyer flasks containing 90 mL of sterile
flask containing 90 mL of sterile distilled water. The flasks were
distilled water. The mixtures were then homogenized by shaking
shaken for 30 min, and 1 mL portions of the solutions were used
for 30 min, and their 1 mL portions were used for serial dilutions
for serial dilutions.
in sterile test tubes.
For each dilution, we used two Petri dishes (90 mm  15 mm)
Two Petri dishes (90 mm  15 mm) containing dichloran 18%
containing dichloran rose bengal chloramphenicol agar (DRBC,
glycerol agar (DG 18, a medium used for analysis of foods with a
recommended for the enumeration of fungi commonly present in
water activity, Aw, below 0.90 (Jarvis et al., 1983) were used
foods; Pitt and Hocking, 1985) and dichloran chloramphenicol
for each dilution. A 0.1 mL aliquot of each sample was then
malt extract agar (DCMA, recommended for the isolation of
transferred to a Petri dish and evenly distributed over its surface
Alternaria species; Andrews, 1992). A 0.1 mL aliquot of each
with a Drigalski spatula. The plates were incubated in an oven at
dilution was added to the Petri dish and evenly distributed over
25 1C for 7 days. The numbers of colony-forming units per gram
the surface with a Drigalski spatula. The plates were incubated in
(CFU/g) were determined thereafter (Pitt et al., 1983). The colonies
an oven at 25 1C for 7 days, and the number of CFU/g was then
were identified to the genus levels according to Pitt and Hocking
determined (Pitt and Hocking, 1997).
(1997).

2.8. Determination of water activity


2.3. Irradiation of seeds and grains
Water activity of the samples was determined with an
The seeds and grains were divided into four groups: Control AQUALAB CX-2 apparatus (Decagon, Pullman, WA, USA).
Group, Groups 1, 2, and 3. Each group consisted of eight 200 g
samples, which were stored separately in plastic bags sealed with 2.9. Scanning electron microscopy (SEM)
adhesive tape and preliminarily irradiated to 20 kGy in order to
eliminate the natural contaminating microbiota. Irradiations were After the incubation, the samples were irradiated, and
carried out at Instituto de Pesquisas Energéticas e Nucleares three seeds or grains from each sample were put into a 40%
(IPEN–CNEN/SP) at temperatures between 25 and 28 1C using a glutaraldehyde solution for 24 h. Next, the seeds or grains were
calibrated cobalt-60 source (Gammacell 220, MDS Nordion, dried at 42 1C for at least 48 h and fixed to appropriate aluminum
Ottawa, Canada) with the dose rate declining from 4.84 to bases. The material was then sputtered with gold, and the sample
4.74 kGy/h. was examined and photographed under a scanning electron
microscope.
2.4. Humidity and temperature control
2.10. Statistical analysis
The samples were stored in sterile receptacles, and Aw was
adjusted to 0.98. A relative humidity 97.5% was maintained with a Statistical analysis of variance of replicate measurements
solution saturated with saline and containing 30% of potassium with the Huynh–Feldt correction was performed with the SPSS
sulfate (K2SO4) (Winston and Bates, 1960). program, Version 12.0. The level of significance was 5%.

2.5. Preparation of the spore suspension 3. Results and discussion

Inoculum of A. alternata (isolated from CATI sunflower seeds 3.1. Fungal mycoflora in unirradiated samples
cultivated at Experimental Station of Zootechny, Nova Odessa)
was added to a Roux flask containing V8 agar (Stevenson, 1974) Fungi were detected in all sunflower seed samples, as well as in
and kept under continuous illumination with cold light for 15 samples of corn, wheat and rice grain, with the genus Aspergillus
days. After this period, the fungal surface was gently scraped with being the most frequent. Fungal counts ranged from 2  104 to
a cell scraper and washed with sterile distilled water and Tween 10  104 CFU/g in sunflower seeds, from 3  104 to 7  104 CFU/g in
80 (2 drops of Tween 80 in 100 mL of the solution). corn grains, from 3  104 to 6  104 CFU/g in wheat grains, and
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Table 1
Numbers of colony-forming units per gram (CFU/g) in the irradiated substrates cultured on DRBC and DCMA.

Substrate CFU/g (  103)

Dose

0 kGy 2 kGy 5 kGy 10 kGy

DRBC DCMA DRBC DCMA DRBC DCMA DRBC DCMA

Sunflower 18.979.7 (8) 10.575.6 (8) 4.472.1 (8) 3.671.8 (8) 0.01 (1) 0.01 (1) – –
Corn 27.3711.6 (8) 25.679.2 (8) 5.873.8 (8) 4.973.7 (8) 0.01 (1) 0.01 (1) – –
Wheat 6.975.4 (8) 3.671.2 (8) 1.770.7 (8) 1.370.6 (8) – 0.01 (1) – –
Rice 9.476.3 (8) 5.374.0 (8) 2.471.8 (8) 1.770.9 (8) – 0.025 (1) – –

The results are reported as means7single standard deviations; the numbers of positive samples are given in the parentheses.

from 2  104 to 105 CFU/g in rice grains. Water activities of the 30


samples were 0.58 for sunflower, 0.75 for corn, 0.70 for wheat, and 0 kGy
0.51 for rice. A. alternata was not isolated, probably due to the low 25 2 kGy
water activity of the analyzed samples. According to Sautour et al. 5 kGy
(2001), the minimal, optimal and maximal Aw levels necessary for 20 10 kGy
the growth of A. alternata are 0.88, 0.98, and 0.99, respectively.
Studies of the occurrence of fungi in sunflower seeds in Brazil 15
are few. Mentem (1985) found 22 potentially pathogenic fungal
species, with A. alternata being one of the most frequent. 10
The importance of A. alternata as a contaminant of sunflower
was confirmed by Pozzi et al. (2005), who detected the fungus in 5
46% of sunflower seed samples, with the highest frequencies
0
observed at water activities from 0.89 to 0.95.
DRBC

DRBC

DRBC

DRBC
DCMA

DCMA

DCMA

DCMA
Aspergillus spp. and Penicillium spp. were isolated from corn
grain samples. The absence of Alternaria spp. and Fusarium spp. in
corn grain might be attributed to the low Aw levels in the corn
samples (0.75), which favor the growth of Aspergillus spp.
Sunflower Corn Wheat Rice
According to Sautour et al. (2001) and Lacey and Magan (1991),
the minimal water activity necessary for the growth of Alternaria
spp. and Fusarium spp. is close to 0.88. In Brazil, several studies Fig. 1. Numbers of colony-forming units per gram (CFU/g) in the irradiated
substrates cultured on DRBC and DCMA.
have found a high frequency of the genera Fusarium, Aspergillus
and Penicillium in corn grains, with Alternaria spp. detected in only
0.2% of the samples (Almeida et al., 2002). In Argentina, González
et al. (1995) frequently isolated A. alternata from corn grains of the For all substrates, the largest number of CFU/g was observed in
1990 harvest. Torres et al. (1998) hypothesized that alternariol the control group (unirradiated) on both the culture media. A
and alternariol monomethyl ether occur in corn naturally. comparison between the control group and the groups irradiated
In our study, the genera Aspergillus and Penicillium were to 2, 5 and 10 kGy showed a reduction in contamination at 2 and
isolated from wheat samples. These results agree with findings 5 kGy and a complete absence of growth at 10 kGy for all the four
of Li and Yoshizawa (2000), who reported a higher frequency substrates. We found fungi (0.1 103 CFU/g) in only one of the
of species of the genera Drechslera, Penicillium, Fusarium, and samples irradiated to 5 kGy. Similar results have been reported by
Aspergillus, among others. In Brazil, Lima et al. (2000) analyzed Ferreira-Castro et al. (2007), who studied the effects of g-radiation
wheat samples stored for 3 months and identified species of the on corn samples artificially contaminated with Fusarium verticil-
genus Alternaria as predominant. Studies conducted in Australia lioides. The authors observed fungal growth in 80% of the samples
have also demonstrated a prevalence of A. alternata and A. infectoria irradiated to 5 kGy and the absence of growth at 10 kGy (the
in wheat samples (Webley et al., 1997). maximal dose used). Aquino et al. (2005), evaluating the effects
Our isolation of genera Aspergillus and Penicillium from the rice of g-radiation on the growth of Aspergillus flavus, demonstrated
grain samples is in line with the findings of Tonon et al. (1997) a higher resistance of the fungus to radiation as compared with
and Nunes (2001). However, A. alternata was the fungus most F. verticillioides and A. alternata, which showed no growth after
frequently isolated from rice grains in Argentina (Broggi et al., exposure to 10 kGy.
2007). According to Aziz et al. (1991), a g-radiation dose of 3 kGy was
sufficient to completely eliminate contamination of tomato juice
with A. alternata; however, Aw was 0.98. In another study using
medicinal plants, Aziz et al. (1997) showed that a dose of 5 kGy
3.2. Fungal mycoflora in irradiated samples was sufficient to eliminate fungi in the samples completely. The
genus Alternaria is known for its resistance to radiation, and some
Fungal contamination of the four substrates studied (sun- Alternaria species survived doses of 4.0 kGy (Beraha et al., 1960;
flower, corn, wheat and rice) was found to decrease with Maity et al., 2004).
increasing g-radiation dose (Table 1, Fig. 1). Water activity was According to Salama et al. (1977), fungi are resistant to
the same (0.98) before and after irradiation in all the substrates. radiation due to mycelial water and the natural radioprotective
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agents. Some investigators postulated that fungi produce numer- Other studies have also shown a higher resistance of dematiac-
ous metabolites, such as alcohols, acids, enzymes, pigments, eous fungi (A. alternata, Cladosporium cladosporioides, Curvularia
polysaccharides, and steroids, as well as some complex com- lunata, and C. geniculata) to g-radiation (Saleh et al., 1988). In a
pounds, such as ergotinine, and antibiotics, including penicillin, subsequent study, Aziz and Moussa (2002) investigated the effects
notatin, flavicin, and fumigacin. In addition, intracellular fungal of g-radiation on the fungal mycoflora of fruits stored at
components (sulfhydric compounds, pigments, amino acids, refrigeration temperatures (below 10 1C) and observed a progres-
proteins and fatty acids) have been reported to be responsible sive reduction of fungal contamination in samples treated with
for radioresistance of fungi (Aziz et al., 1997; Silveira, 1995). 1.5 and 3.5 kGy. Ladaniya et al. (2003), who exposed three citrus
Melanin, a polymer that protects live organisms against UV rays species to low g-radiation doses (0.25, 0.5, 1, and 1.5 kGy) and then
and ionizing radiation, has also been associated with fungal stored them at 6–7 1C for 75–90 days, showed that the dose
radioresistance, especially among dematiaceous fungi. Aquino of 1.5 kGy was not sufficient to completely control fungi: it only
(2007), analyzing medicinal plant samples, demonstrated a delayed fungal growth and, thus, increased the shelf-life of the
higher resistance of Phoma spp. to a radiation dose of 5 kGy. fruits.

Intact A. alternata with spores in V8 medium

0 kGy 2 kGy

5 kGy 10 kGy
Sunflower

Fig. 2. SEM images of the cultures.


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0 kGy 2 kGy

5 kGy 10 kGy
Corn

0 kGy 2 kGy

5 kGy 10 kGy
Rice

Fig. 2. (Continued)

According to the World Health Organization (1994), a dose of of fungi in foods (Saleh and Aziz, 1996; Abd El-Aal and Aziz, 1997).
2 kGy markedly reduces the number of microorganisms present in These findings agree with the results of our study, which revealed
foods, and higher doses (4–6 kGy) completely inhibit the presence fungi in samples treated with a dose up to 5 kGy.
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0 kGy 2 kGy

5 kGy 10 kGy.
Wheat

Fig. 2. (Continued)

ANOVA and the Huynh–Feldt correction test (po0.05) showed contrast, twisted filamentous forms with marked alterations in
a statistically significant difference in the fungal counts between the shape and surface of the hyphae and an aspect of ‘‘dehydra-
the unirradiated samples and the samples irradiated to 2 kGy, for tion’’ and ‘‘rupture of the filaments’’ were found in the samples
all the substrates. There was no statistically significant difference exposed to 5 and 10 kGy. Filamentous forms featuring a melting-
between the counts in the two culture media (DRBC and DCMA) like aspect and apparent adhesion to the seed surface were
for the unirradiated and irradiated (2 kGy) samples of corn, rice observed in sunflower seeds treated identically, probably due to
and wheat (p40.05); however, there was a significant difference rancification. However, the numbers of intact hyphae in corn, rice
for sunflower (po0.05). and wheat grains were smaller. There were no spores in
inoculated grains. Ferreira-Castro et al. (2007) observed similar
alterations increasing with dose.
3.2.1. SEM
The advantages of SEM, as compared with light microscopy, are
a better resolution, higher magnification, greater depth of field
and greater versatility (Goodhew and Humpreys, 1998). It makes 4. Conclusions
fungal structures on the substrate and fungal growth more visible
(Bacon et al., 1992). SEM studies conducted by Torres et al. (2003) g-Irradiation to 5 kGy was effective in slowing the growth of
have shown that water activity and temperature affect the growth A. alternata. However, a dose of 10 kGy was necessary to inhibit
of Aspergillus ochraceus, A. alternata and Fusarium verticillioides in fungal growth completely. DRBC medium was more effective in
maize grains. Murillo et al. (1999) used SEM to observe hyphal the isolation of A. alternata in sunflower seeds. SEM made it
penetration of F. moniliforme in maize grains (Fig. 2). possible to identify structural changes induced by irradiation
In this study, we analyzed the samples irradiated to 2, 5, and to the different doses, which confirmed the CFU counting results.
10 kGy, as well as control samples, with SEM in order to evaluate
morphological changes resulted from the exposure to ionizing
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